Much effort has been devoted in recent years to the production of biofuels, such as ethanol, butanol, higher-chain alcohols, and hydrocarbons via microbial fermentation of sugars and other biomass constituents.
To date, the fatty acid biosynthesis pathway has been widely used as the means to generate higher-chain (C≥4) acyl-CoA thioesters required for the synthesis of the aforementioned products. However, the operation of this pathway is not efficient because it consumes ATP in the synthesis of malonyl-ACP, which is the donor of two-carbon units for chain elongation. As a consequence, the ATP yield associated with the production of hydrocarbon through the fatty acid synthesis pathway is very low. This, in turn, greatly limits cell growth and hydrocarbon and other product production.
We have implemented an entirely novel approach, driving beta oxidation in reverse to make fatty acids instead of degrading them (see US20130316413, WO2013036812, each incorporated by reference in its entirety for all purposes). Unlike the fatty acid biosynthesis pathway, the reversal of the β-oxidation cycle operates with coenzyme-A (CoA) thioester intermediates and uses acetyl-CoA directly for acyl-chain elongation (rather than first requiring ATP-dependent activation to malonyl-CoA).
A engineered microorganism having a reverse beta oxidation cycle that produces alcohols, carboxylic acids, and hydrocarbons, and derivatives thereof, generally includes i) expression of the β-oxidation cycle in the absence of fatty acids and presence of a non-fatty acid carbon source, ii) functional operation of the β-oxidation cycle in the reverse or biosynthetic direction (e.g. making fats rather than degrading them), iii) overexpression of one or more termination enzymes that convert reverse beta oxidation cycle intermediates to a desired alcohol, carboxylic acid, or hydrocarbon, thus exiting or terminating the cycle for that intermediate. Further, any of the alcohols, carboxylic acids, and hydrocarbon products can be further modified to make other products, such as aldehydes, and the like, in secondary termination pathways.
The utility of this technology relates to the efficient synthesis of hydrocarbons, etc. using an engineered reversal of the β-oxidation cycle, which in turn will establish a new paradigm for the production of advanced biofuels. The ubiquitous nature of β-oxidation enzymes should enable the combinatorial synthesis of non-native products in industrial organisms with a minimum number of foreign genes, an approach that would ensure the efficient functioning of the engineered pathways. By enabling the production of products through a functional reversal of the β-oxidation cycle, this technology will contribute to the creation of fundamentally new approaches that could enable efficient production of second-generation biofuels.
We take the above research forward in this disclosure, adding further diversification of enzymes to be used as part of this pathway.